The movement of N-arachidonoylethanolamine (anandamide) across cellular membranes

Abstract

This review presents and explores the hypothesis that N-arachidonoylethanolamine (AEA, also called anandamide) is transported across cellular membranes by a process that is protein-mediated. Support for this hypothesis comes from experiments demonstrating that cellular accumulation of extracellularly applied AEA is saturable, time and temperature dependent and exhibits selective inhibition by various structural analogs of AEA. The accumulation of AEA is cell specific; data is presented demonstrating that several cell types, including the bovine adrenal zona glomerulosa cell, exhibit very high capacity for AEA accumulation while others, such as the HeLa cell, have a very low capacity. The transport process has the characteristics of facilitated diffusion; it is bi-directional, not dependent on either ATP or extracellular sodium and exhibits the trans effect of flux coupling. Several important questions remain to be answered regarding the carrier, including its molecular structure and its role in the release and inactivation of endogenously produced AEA.

Introduction

The evidence supporting a role for the endocannabinoid N-arachidonoylethanolamine (AEA or anandamide) as an intercellular signaling molecule is mounting. AEA is present in measurable amounts in the brain (Devane et al., 1992, Schmid et al., 1995, Felder et al., 1996, Schmid et al., 1996), uterus (Schmid et al., 1997) and testes (Sugiura et al., 1996, Kondo et al., 1998). AEA binds and activates the CB1 cannabinoid receptor (Devane et al., 1992, Felder et al., 1993, Fride and Mechoulam, 1993) and mimics the physiological and biochemical effects of the classical cannabinoids (see Hillard and Campbell, 1997 for review). In the rodent brain, there is a reasonably good match between the regional distribution of AEA or its precursor, N-arachidonoylphosphatidylethanolamine (N-arachPE) (Bisogno et al., 1999, Yang et al., 1999) and the distribution of the CB1 cannabinoid receptor (Tsou et al., 1998a, Herkenham et al., 1990). The cellular synthesis of AEA has been demonstrated in neurons in primary culture (Di Marzo et al., 1994, Cadas et al., 1996); human umbilical vein endothelial cells (HUVECs) (Maccarrone et al., 2000); neuroblastoma cells (Di Marzo et al., 1996); macrophages and macrophage-derived cell lines (Di Marzo et al., 1996, Bisogno et al., 1997, Pestonjamasp and Burstein, 1998, Varga et al., 1998) and basophilic cells (Bisogno et al., 1997, Rakhshan et al., 2000).

Among the criteria for the classification of an intercellular signaling molecule or transmitter is evidence of a process by which its signaling is terminated. The actions of most transmitters are terminated by (1) diffusion away from its site of action; (2) metabolism to one or more inactive compounds; (3) transport to an intracellular compartment where the transmitter is sequestered from its site of action; or (4) a combination of these processes.

There is considerable evidence that AEA is metabolized to molecules that do not activate the CB1 cannabinoid receptor. AEA is a substrate for an amidohydrolase which converts AEA to arachidonic acid (which is rapidly reesterified into cellular phospholipids) and ethanolamine (Deutsch and Chin, 1993). The AEA amidohydrolase activity is identical to that of a cloned fatty acid amide hydrolase (FAAH; Cravatt et al., 1996) which also catabolizes the putative sleep inducing lipid oleamide (Cravatt et al., 1995). AEA amidohydrolase is a membrane protein whose distribution in cellular membrane fractions correlates with the distribution of markers for endoplasmic reticular membranes (Hillard et al., 1995, Ueda et al., 1995). Conversely, membrane fractions from rat brain that are enriched in plasma membranes have very low AEA amidohydrolase activity (Hillard et al., 1995). Immunohistochemical data support an intracellular localization for AEA amidohydrolase in brain (Tsou et al., 1998b). Therefore, if AEA amidohydrolase plays a role in the inactivation of AEA that has been released, then a process must be present for the movement of AEA from the extracellular space into cells that contain AEA amidohydrolase.

The subject of this review is the evidence that supports the hypothesis that AEA transport across cellular membranes occurs via a protein-mediated mechanism. One possible function of this transport process is to act in series with intracellular AEA amidohydrolase to inactivate extracellular AEA. Another function of the transport process may be to sequester and functionally inactivate AEA by removing it from its receptors. Finally, it is also possible that a transport process serves to release AEA from cells after its synthesis, implying a role for an AEA transport protein in the process of activation of the AEA/cannabinoid signaling system.